The present invention relates to the entire gene encoding .beta.3-adrenergic receptor polypeptides of mouse and human origin, which polypeptides provide a procedure for studying the effects of various chemical agents on the .beta.3-adrenergic receptor and coupled adenylate cyclase and hormone-sensitive lipases. More particularly, the present invention also relates to antibodies, vectors, nucleotide probes, cell hosts transformed by genes encoding polypeptides, having .beta.3-adrenergic receptor activity.
In the past, two main classes of adrenergic receptors have been identified as the a adrenergic receptors and the .beta. adrenergic receptors. These adrenergic receptors produce various responses to effector organs such as the eye, heart, arteriols, veins, lungs, stomach, intestine, gallbladder, kidney, skin, spleen, liver and pancrease to mention a few. These specific receptors have been defined by the effects of particular synthetic agonists which stimulate the receptors biological function and antagonists which block the adrenergic receptors biological function.
Within the two classes of adrenergic receptors, four subtypes, .alpha.1, .alpha.2, .beta.1 and .beta.2, of these receptors for catecholamines have been identified. See, Cotecchia et al., P.N.A.S., 85, pgs. 7159-7163 (1988); Kobilka et al., Science 238, pgs. 650-656 (1987); Frielle et al., P.N.A.S., 84, pgs. 7920-7924 (1987); and Emorine et al., P.N.A.S., 84, pgs. 6995-6999 (1987). Drugs that selectively block or stimulate one of these receptor subtypes are used extensively in clinical medicine. Despite the efficacy of these drugs, many produce side effects in individuals, due to their interaction with other receptor subtypes. For identification of the various drugs which act on the receptor subtypes, see Goodman and Gilman's The Pharmacological Basis of Therapeutics, eigth edition, MacMillan Publishing Co., Inc. (1990).
The analysis of the genes of these receptors indicate that all of the subtypes of the receptors belong to the family of integral membrane receptors exhibiting striking homologies, in particular these homologies are present in the 7 transmembrane regions. These receptors are coupled to regulatory proteins termed G proteins which are capable of binding molecules of guanosine triphosphate (GTP).
The G proteins have the capacity to intervene structurally and functionally between receptors and enzymes catalyzing the production of intracellular mediators such as adenylate cyclase, guanylate cyclase, the phospholipases and the kinases, or between receptors and ion channels. Thus, the G proteins have the capacity to regulate the flux of ions such as calcium, potassium, sodium and hydrogen ions.
The above-mentioned subtypes of receptors are known in the art as the "R.sub.7 G family" as described by Emorine et al. Proc. NATO Adv. Res. Workshop (1988). The R.sub.7 family comprises acetylcholine muscarinic receptors, serotonin receptors, the receptors for neuropeptides, substance K, angiotensin II and the visual receptors for the opsins, as described by Dixon et al Annual Reports in Medicinal Chemistry, pgs. 221-233 Ed. Seamon, K. B., Food and Drug Administration, Bethesda, Md. (1988) and Emorine et al., supra.
Recently, a third subtype of the .beta.-adrenergic receptor termed .beta.3-adrenergic receptor, has been identified and characterized in humans and in rodents, which polypeptide does not have a receptor activity similar to that of the .beta.1- or .beta.2-adrenergic receptors. This "new" .beta.3-receptor in humans has been identified and sequenced as described in French application No.8900918 filed Jan. 25, 1989 and PCT/FR90/00054, resulting in U.S. application Ser. No.07/721,571, filed Jan. 25, 1990, U.S. Pat. No. 5,288,607, incorporated herein by reference, as containing 402 amino acids, which is capable of activating adenylate cyclase in the presence of an agonist, which activity increases in the order of agonists of salbutamol, BRL 28410, BRL 37344 and (1)-isoproterenol. Antibodies directed to the polypeptide of the .beta.3-adrenergic receptor were also disclosed. The .beta.3-receptor was identified as differing from the .beta.1-adrenergic receptor and the .beta.2-adrenergic receptor by a pharmacological comparison of the activation of adenylate cyclase in the presence of agonists and the reaction towards different antagonists.
Similarly, the .beta.3-adrenergic receptor of the mouse has been identified and cloned as described in French application No. 9100320 filed Jan. 14, 1991 and PCT/FR92/00023 filed Jan. 15, 1992, also incorporated herein by reference. See also, Nahmias et al., Embo J., 10, pgs. 3721-3727 (1991). The mouse .beta.3-adrenergic receptor encodes a polypeptide of 388 amino acid residues, including the features characteristic of .beta.-adrenergic receptors, such as the conserved amino acids identified as crucial for catecholamine binding in the .beta.2-adrenergic receptor. See, Strader et al., FASEB J. 3, pgs. 1825-1832 (1989).
A pharmacological comparison of the mouse with the human counter part of the .beta.3-adrenergic receptors indicates that these receptors have similar reactivity. For example, mouse .beta.3-adrenergic receptor can be activated by CGP 12177A, oxprenolol and pindolol while displaying a low stereoselectivity and characteristic potency order for full agonists, which is similar to the human.
However, differences do exist between the mouse .beta.3-adrenergic receptor and the human .beta.3-adrenergic receptor, which are probably due to the structural differences observed between these two receptors in the transmembrane domains which are involved in ligand binding wherein 12 substitutions do occur.
Thermogenesis and lipolysis in brown and white adipose tissues are under the control of this .beta.3-adrenergic receptor subtype as described by Arch et al., Proc. Nutr. Sci., 48, pgs. 215-223 (1989); Arch et al., In Obesity, John Wiley & Sons Ltd., London (1991); and Zaagsma et al., Trends Pharmacol. Sci., 11, pgs. 3-7 (1990). Besides adipose tissues, the expression of the .beta.3-adrenergic receptor has also been reported in various other tissues such as tissues of the digestive tract, from oesophagus to colon and in the gallbladder. See, for example, Bond et al., Br. J. Pharmacol., 95, pgs. 723-734 (1988); Coleman et al., British Journal of Pharmacology Proc. Supl., 90, 40 (1987); Bianchetti et al., Br. J. Pharmacol., 100, pgs. 831-839 (1990); Granneman et al., J. Pharmacol. Exp. Ther., 256, pgs. 421-425 (1991); and Krief et al., J. Clin. Invest., (1993) (in press). This .beta.3-adrenergic receptor subtype has been suggested to participate in the control by catecholamines of body energy balance from intestine assimilation to storage and mobilization in adipose tissue.
Factors such as temperature, feeding or fasting and stress influence the body's hormonal status, thus inducing tissue specific adaptive modifications of energy balance which may in part result from regulation of cellular .beta.3-adrenergic receptor sensitivity. It has been further shown that .beta.-adrenergic agonists, glucocorticoids and several other agents can modulate .beta.3-adrenergic receptor density, responsiveness and mRNA levels. The molecular determinants responsible for the regulation of cellular .beta.3-adrenergic receptor may possibly be found either on the receptor itself or in its gene or mRNA. For example, post-translational modifications of the receptor in the third intra-cytoplasmic loop or in the carboxy-terminal tail may modulate .beta.-adrenergic receptor coupling to adenylate cyclase. Factors which act on .beta.-adrenergic receptor gene transcription rate or on mRNA stability may also modulate cellular adrenergic responsiveness by modifying .beta.-adrenergic receptor expression levels.
The .beta.3-adrenergic receptors have been sequenced and identified, as well as compared pharmacologically with other known receptors in mouse and humans. A comparison of the .beta.3-adrenergic receptor amino acid sequences predicted from the nucleotide sequences of the human and mouse genomic genes revealed differences in the carboxy-terminal regions of the receptors. Although these differences could be attributed to evolutionary species-related variations, it was recently discovered by the present inventors that the entire coding sequence for the previously reported .beta.3-adrenergic receptor in mouse and human was not complete. The genes encoding .beta.1- and .beta.2- comprise one exon and it was thought that the .beta.3-adrenergic receptor gene, when initially identified, also comprised only one exon. However, it was recently and unexpectedly discovered that contrary to .beta.1- and .beta.2-adrenergic receptor genes, the human and mouse .beta.3-adrenergic receptor genes comprise several exons. The identification of the introns and exons in mouse and human .beta.3-adrenergic receptors and thus the entire gene itself, permits a full characterization of this receptor which will aid in more sensitive genetically engineered products such as nucleotide probes, polyclonal and monoclonal antibodies and the like for drug testing and diagnostic purposes, and permits the understanding of .beta.3-adrenergic receptor regulated expression in various tissues.